The present invention relates to the identification of Bacteroides gingivalis in biological samples by detection of a specific peptidase activity of B. gingivalis. The detection is suitably carried out by a colorimetric analysis of enzymes uniquely provided by B. gingivalis.
Certain species of gram negative bacteria, such as B. gingivalis, have been implicated in the etiology and pathogenesis of certain forms of periodontal disease. Such pathogenic bacteria secrete enzymes which are strongly suspect in pathogenesis of periodontal infections. For example, proteolytic enzymes such as collagenase, neuraminidase, fibrinolysin, trypsin like enzymes, and aminopeptidases are produced by various oral microorganisms and are closely associated with periodontal destruction.
Clinical assays specific to B. gingivalis in gingival and subgingival dental plaque are particularly useful in the diagnosis of periodontal disease, in evaluating the progress of periodontal therapy, and in determining the status of the patient at recall examinations. The standard bacteriological techniques presently in use to identify such microorganisms are time consuming, expensive, and require a high level of technical expertise. Further, such tests frequently give results which are not as accurate or as sensitive as may be desired or required.
Previously, studies have been carried out in attempts to identify B. gingivalis by its proteolytic activity. Much of this work has been done to specifically identify enzymes which are strongly associated with periodontal disease. However, many of the enzymes strongly associated with periodontal disease are not singularly specific to B. gingivalis. Examples of prior art studies are:
1. Yoshimura et al., Archives of Oral Biology, 1984, reports a trypsin-like membrane bound protease from B. gingivalis which hydrolyzes benzoyl-L-arginine-p-nitroanilide (L-BAPA), benzoyl-DL-arginine-.beta.-naphthylamide (BANA) and tosyl-L-arginine methyl ester. This enzyme activity is not specific to B. gingivalis. The same enzyme activity is found, for example, in Treponema denticola.
2. Abiko et al.; Journal of Dental Research, 1985, reports the purification an enzyme from B. gingivalis which degrades dipeptidyl substrates. This enzyme is not specific to B. gingivalis. It is also found in Capnocytophaga.
3. Dellinger and Moore, Journal of Clinical Microbiology, 1986, report the use of a commercially available system, RapID-ANA, for the identification of bacterial species. This system consists of a number of protein and sugar substrates and the bacteria are identified by their pattern of reactivity. B. gingivalis may be identified, but only through a series of separate eliminations steps.
4. Laughon et al., Journal of Clinical Microbiology, 1982, reports the use of a commercially available system, API-ZYM, for the identification of oral bacterial species. The substrates are protein or sugars. A common trypsin-like activity was found in B. gingivalis, T. denticola, and Capnocytophaga, however, the activity is not specific for B. gingivalis.
The present invention relates to the selective, singular detection of B. gingivalis by detection of a bacterial enzyme specific to B. gingivalis. Bacterial enzymes may be classified as either constitutive or adaptive. Constitutive enzymes are those enzymes which are formed by the bacterial cell wall under substantially all conditions of growth. In contrast, adaptive enzymes are those enzymes formed by the bacterial cell only in response to an inducer. Adaptive enzymes allow the cell to control to some extent the environment in which the cell lives. In nature the inducer is usually the substrate for the specific adaptive enzyme involved. Bacterial enzymes may also be divided into enzymes remaining in the cell (endoenzymes) and enzymes secreted by the cell into the surrounding medium (exoenzymes).
The enzymes utilized in the present invention are both adaptive and endoenzymes.